Apoptosis is a cellular process wherein cells initiate a series of events that lead to their ultimate demise. Apoptosis is as important to cell life cycle regulation as growth processes such as mitosis. Normal cells use apoptosis to insure appropriate development and protect against anything that may threaten cell integrity. Abnormal cells lose the ability to properly regulate themselves through apoptosis. When there is not enough apoptosis, cells will grow out of control as demonstrated in cancerous disease states. In other diseases, cells may exhibit too much apoptosis. For example, the decline of CD4+ T cells resulting from AIDS is likely due to apoptosis.
There are three mechanisms by which cells may initiate apoptosis: intrinsic pathway, extrinsic pathway, and apoptosis inducing factor (AIF). In the intrinsic pathway, also known as the mitochondrial pathway, the apoptotic process is triggered by internal cellular signals. Conversely, the extrinsic pathway, also called the death receptor pathway, is due to an external signaling mechanism. Independent of the intrinsic and extrinsic pathways, some cells require a specific protein, AIF, to trigger apoptosis.
Regardless of the mechanism by which apoptosis occurs, there are a series of morphological changes that are detectable and considered to be the standard to define the mode of cell death. Some of the changes include cell shrinkage, cell surface blebbing, nuclear chromatin condensation, and apoptotic body formation. Typically, a trained eye, using simple techniques such as microscopy, cytometry, and imaging, detects the morphological changes.
The morphological changes of cells during the apoptotic process are due to underlying biochemical and molecular events. The biochemical and molecular events are more difficult to ascertain because they typically require complex cellular assays that are usually tedious, unreliable, or lead to results that are difficult to interpret. For example, annexin V labeling is a common cellular assay to confirm apoptosis. Apoptotic cells lose their ability to regulate the composition of their lipid membranes and phosphatidylserine (PS), which is located on the internal plasma membrane in normal cells, is externalized and expressed on the outer plasma membrane of apoptotic cells. Annexin V is a protein that binds to PS and is used as a fluorescent marker to label PS in several commercially available apoptosis assay kits. Although annexin V labeling is a current method to detect apoptosis, challenges remain in the interpretation of the results from this type of assay.
There exists a need to be able to detect the biochemical changes in cells without a reagent-based, multi-step cellular assay. The present disclosure provides such a method using Raman spectroscopy.